The objective of this research program is to use a combination of molecular genetics, protein biochemistry, global gene expression profiling, proteomics and bioinformatics approaches to elucidate the molecular mechanism of the SmpB.SsrA ribosome rescue system and its role in survival, virulence, and pathogenesis of two category A pathogens, Yersinia pestis and Francisella tularensis. The menace of bioterrorism and the emergence of antibiotic resistance in an increasing number of pathogenic bacterial highlight the need for the development of novel classes of anti-infective agents. Existing antimicrobial drugs target known cellular mechanisms associated with protein synthesis, DNA replication, cell wall and membrane synthesis, cell division, or nutrient uptake and transport. New antibiotic development needs to focus on novel targets and will require a basic molecular understanding of their cellular function. Promisingly, bacteria possess a unique target -the SmpB.SsrA mediated ribosome rescue, protein tagging and directed degradation system- that is thought to participate in the adaptation and survival of pathogenic bacteria in the hostile micro-niches of their hosts. This unique system, also known as trans-translation, is orchestrated by a remarkable RNA (SsrA RNA) that functions both as a tRNA and an mRNA to facilitate the addition of a short degradation tag to the C-terminus of nascent polypeptides. All known activities of SsrA require SmpB, a small protein that binds SsrA specifically and with high affinity to promote its association with stalled ribosomes. The molecular basis for the formation of the SmpB.SsrA complex, its recognition of impaired ribosomes, and the subsequent addition of the degradation tag are not well understood. To assist us in these studies, we will develop an in vitro trans-translation system that should enable us to scrutinize the mechanistic details of the SmpB-SsrA mediated ribosome rescue process. This in vitro system will also be utilized as a screening assay for selection of small molecules that specifically inhibit this process. Furthermore, we wish to identify and characterize additional cellular factors that participate in this process for use as novel targets. Because the trans-translation system exists exclusively in prokaryotes and involves novel RNA and protein factors that are essential for the survival of most (if not all) pathogenic bacteria, a better understanding of this unique process should permit the discovery and/or design of highly specific novel anti-bacterial agents.